The metabolic product of methylazoxymethanol (MAM) responsible for tumorigenesis appears to be the corresponding aldehydic derivative. This is based on the fact that MAM is substrate in NAD(plus)-dependent dehydrogenase reactions in vitro and on the fact that in vivo, disulfiram, an inhibitor of aldehyde dehydrogenase, potentiated the biological effects of MAM. The major aim of this proposal is to determine whether the product is, in fact, the proposed aldehyde and, if this should be so, whether it functions as a bifunctional alkylating agent or whether it is unstable and, as a result of its spontaneous decomposition, gives rise to carbonium ions at a rate faster than that which occurs from the spontaneous decomposition of MAM. We propose to incubate either guanine-8-14C or cysteine-1-14C with MAM alone and also with MAM plus NAD plus alcohol dehydrogenase. The amount of MAM metabolized can be monitored at 340 millimicrons which is an indicator of the amount of NADH formed. The amounts of the alkylated compounds will be determined by separation on column chromatography. We will also incubate either nucleosides or calf thymus DNA. Crosslinked nucleotide products can be determined by HPLC, and crosslinking of DNA will be determined from UV-absorbance measurements. We plan to identify the activating enzyme(s) in different organs sensitive to MAM. Affinity chromatography can be used for separation of individual NAD(plus)- and NADP(plus)-dependent dehydrogenases. Dimethylhydrazine (DMH) is metabolized to MAM and the MAM is excreted in the urine. To obtain 14C-MAM, we have treated rats with 14C-DMH and will collect the 14C-MAM. We will study the physiological disposition of MAM and determine the products of its reaction with nucleic acids and protein.